摘要
目的:研制一种检测β胶联降解产物(β-CTX)和骨钙素N端中分子片段(N-MID)水平的双标记时间分辨免疫荧光分析方法(TRFIA)并评价其检测性能。方法:将抗β-CTX和N-MID的4E5和2B7单克隆抗体(MAb)作为包被抗体包被在96孔培养板,采用铕离子(Eu^(3+))和钐离子(Sm^(3+))分别标记2G6和5A3 MAb作为检测抗体,建立双抗体夹心TRFIA分析法并制备成试剂盒。通过试剂盒的灵敏度、准确度(稀释回收率)、特异性、精密度、稳定性和临床样本比对等实验评价其检测性能。结果:制备的双标记TRFIA试剂盒对β-CTX的检测灵敏度为0.025μg·L^(-1),线性范围为0.025~5.000μg·L^(-1),对N-MID的检测灵敏度为0.5μg·L^(-1),线性范围为0.5~200.0μg·L^(-1)。β-CTX平均稀释回收率为102.13%,N-MID平均稀释回收率为103.02%,与其他常见骨检测指标无明显交叉反应,特异性较强。β-CTX批内变异系数(CV)为5.81%~7.82%,批间CV为5.97%~8.02%;N-MID批内CV为6.05%~8.32%,批间CV为6.14%~8.56%;TRFIA试剂盒可在4℃稳定保存6个月,37℃稳定保存7 d。结论:建立的双标记TRFIA方法具有高灵敏度、高特异度、高准确率和方便快捷等优点,适用于大批量临床样品的检测。
Objective:To develop a double-labeled time-resolved immunofluorescence analysis(TRFIA)method for the determination of the levels ofβ-cross-linked C-terminal telopeptide of typeⅠcollagen(β-CTX)and N-terminal middle molecular fragment of osteocalein(N-MID)of osteocalcin,and to evaluate its detection performance.Methods:The 4E5和2B7 monoclonal antibodies(MAb)ofβ-CTX and N-MID antigens were constructed,and then coated as coating antibodies in 96-well plates,and the antibodies were labeled with europium ion(Eu^(3+))and samarium ion(Sm^(3+)),respectively.A double antibody sandwich TRFIA method was established and prepared into a kit.The detection performance was evaluated by the sensitivity,accuracy(dilution recovery),specificity,precision,stability,and clinica sample alignment of the kit.Results:The prepared double-labeled TRFIA Kit had a detection sensitivity of0.025μg·L^(-1) forβ-CTX and a linear range of 0.025-5.000μg·L^(-1);the detection sensitivity for N-MID was 0.5μg·L^(-1),and the linear range was 0.5-200.0μg·L^(-1);the average dilution recovery rate ofβ-CTX was 102.13%,and the average dilution recovery rate of N-MID was 103.02%;there were no obvious cross-reaction with other common bone test indicators and the specificity was high.The intra-assay coefficient of variation(CV)ofβ-CTX was 5.81%-7.82%,and the inter-assay CV was 5.97%-8.02%;the intra-assay CV of N-MID was 6.05%-8.32%,and the inter-assay CV was 6.14%-8.56%;the TRFIA Kit could be stored stably at 4℃for half a year and stably stored at 37℃for 7 d.Conclusion:The establishedβ-CTX and N-MID double-labeled TRFIA method has the advantages of high sensitivity,high specificity,high accuracy,convenience and speed,and is suitable for the detection of largescale clinical samples.
作者
毛骞
陈翠翠
梁焕坤
刘鹏娥
钟树海
李来庆
MAO Qian;CHEN Cuicui;LIANG Huankun;LIU Penge;ZHONG Shuhai;LI Laiqing(Department of Endocrinology,Affiliated Hospital,Beihua University,Jilin 132001,China;Guangzhou Youdi Biotechnology Co.,Ltd.,Guangzhou 510663,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2021年第3期770-776,共7页
Journal of Jilin University：Medicine Edition
基金
吉林省卫计委卫生技术创新项目(2017J076)。
关键词
β胶联降解产物
N端中分子片段
骨质疏松症
双抗体夹心
双标记时间分辨免疫荧光分析
β-cross-linked C-terminal telopeptide of typeⅠcollagen
N-terminal middle molecular fragment of osteocalein
osteoporosis
double antibody sandwich
double-labeled time-resolved immunofluorescence analysis
